AMER. ZOOL., 13:557-563 (1973).
Acid-Base Regulation and Temperature in Selected Invertebrates as a
Function of Temperature
B. J. HOWELL, H. RAHN, D. GOODFELLOW, AND C. HERREID
Department of Physiology, Department of Biology, State University of New York
at Buffalo, Buffalo, New York 14214
SYNOPSIS. The pH of the hemolymph of selected invertebrates decreases as their body
temperature increases. The magnitude of this change (A£H/A°C) is very similar to
the change of the pH of water with temperature (ApiV/A°C) and suggests that
these invertebrates, like poikilothermous vertebrates, regulate the pH of their extracellular fluid so that its degree of alkalinity relative to the pH of water remains constant. The degree of alkalinity (pHblooa-£2V) varies between species, but seems to be
fixed for any given species. In Limulus pH-pN was essentially the same for in vivo
samples, measured after the whole animal had been acclimated to different temperatures, as it was for in vitro samples in which the hemolymph was cooled or
warmed anaerobically, suggesting that the CO2 content of the extracellular fluid
is constant as the temperature changes. The Poc>2 of the hemolymph is invariably
lower in animals breathing water than in those breathing air. In the invertebrates,
as in the vertebrates, manipulation of PCo2 and HCO3- is probably the major mechanism in the regulation of the relative alkalinity of the extracellular fluid.
At their normal body temperature homeotherms maintain the pH of their blood
at a fixed value by altering the plasma
H2CO3 by ventilation and/or adjusting the
plasma HCO3~ by renal regulation. Poikilotherms, on the other hand, do not have
a constant body temperature nor a fixed
value for the pH of their extracellular
fluids.
Austin and Cullen (1925) analyzed theoretically and in vitro the effect of temperature variations on the reaction of blood
and pointed out, for the first time to our
knowledge, that in aqueous solutions such
as blood, one must consider not only
changes in H+ but also changes in OH~
since the concentrations of both of these
ions in water are altered as the temperature changes. They suggested that in biological studies dealing with the effect of
temperature on blood reaction, the ratio
of H+ to OH~ might be the significant
factor rather than the absolute concentration of the hydrogen ion. In 1927, Austin
et al. tested this hypothesis on unanestheSupported in part by O.N.R. Contract No.
N00014-68-A-0216, and Alpha Helix, Bering Sea
Program, 1968.
Present address of D. Goodfellow: School of Nursing, University of Tennessee, Knoxville, Tennessee
37916.
557
tized alligators and showed that, indeed,
between the temperatures of 9 and 35 C,
the blood of the alligator maintained a constant H + / O H - .
Little attention was paid to that early
work on poikilotherms until Rahn (19666)
suggested that the regulation of acid-base
balance in cold-blooded vertebrates was
centered around the maintenance of a constant relative alkalinity of the blood.
The concept of relative alkalinity
The fact that the pH of distilled water
is 7.0 only at 25 C has long been known but
largely ignored by biological scientists. As
temperature increases, the ionization constant of water, Kw, increases; the negative logarithm of Kw, pKw, thus decreases.
pKw = pH -J- pOH and since water is
a neutral substance, pH = pOH at all temperatures. The pKw varies from 14.734 at
5 C to 13.620 at 37 C while the pYL and
pOH vary from 7.367 at 5 C to 6.810 at
37 C. The pU or pOH of water at any
temperature is equal to one-half pKw and
since water is neutral, we refer to this
value, i/2 pKw, as pN as Austin and
Cullen (1925) suggested. The change in
the neutrality of water (pN).as a function
of temperature is plotted at the bottom
of Figure 1. The shaded band above this
558
H O W E L I , RAHN, GOODFELLOW, AND HERREID
METHODS
FIG. 1. The top band represents the blood pH of
725 vertebrates as a function of body temperature.
The lower line represents pN, the pH, or pOH of
water, at the given temperature. (From Rahn,
1971.)
line represents the blood pH of normal,
unanesthetized amphibians and reptiles
(Howeli et al., 1970) and fish (Rahn and
Baumgardner, 1972). Included in this band,
at the higher temperature range, are values
for homeotherms measured at their normal
body temperature. The available data,
some 725 blood pH values for both coldblooded and warm-blooded vertebrates,
have recently been presented by Baumgardner (1971) and Rahn (1971).
The decrease in the pH of vertebrate
blood
with
increasing
temperature
(ApH/A°C = —0.016) is essentially the
same as the change in pN (ApAf/AoC =
—0.017). It was this apparently close relationship between the change in the pH of
the blood and the change in the pK of
neutral water that led Rahn (1966b) to propose that cold-blooded vertebrates regulated the hydrogen ion concentration in
such a way that their circulating fluids
were always maintained relatively alkaline
to water. Thus, the pH of the blood at any
temperature is always maintained 0.6-0.8
pH units higher than pN. This value, pHpN, varies between species, but seems to
be a constant for any given species over
their viable temperature range.
The possibility that acid-base regulation
in all poikilotherms might be related to
the maintenance of a constant relative
alkalinity led us to test the hypothesis in
selected invertebrates.
Cnllinectes sapidus, Carcinus macnas,
Uca pugilator, and Limuhis polyphemus
were obtained from Woods Hole, Massachusetts. Gecarcinus lateralis was supplied
by Dr. Dorothy Bliss of the American Museum of Natural History. Callinectes, Carcinus, and Limulus were maintained in artificial sea water; Gecarcinus and Uca were
kept on moist sand in air. Data from the
Alaskan King Crab (Paralithodes camtschaticus) had been obtained in 1968 during an expedition of the Alpha Helix off
Kodiak, Alaska. All animals were unfed
during the experimental period.
A group of animals was maintained at
a given temperature for at least one week.
On the day of sampling the microelectrode
temperature was set at the temperature of
the animal's environment and calibrated
at that temperature with two precision
buffers. Hemolymph was withdrawn anaerobically into a syringe in which the
dead space had been filled with Heparin.
Duplicate determinations of the pH were
immediately made on a portion of the
sample. The remainder of the fluid was
used to determine the PC02 of the hemolymph by the Astrup method (1956). The
animals were then placed at a different
temperature and maintained at that temperature for at least a week. Then, the
pH. and PC02 were determined at the new
temperature. Values of pH and PC02 were
measured at at least three different temperatures. It was occasionally necessary to
combine samples from two of the Uca in
order to get enough fluid to make the Pco»
measurements.
For the in vitro study reported for
Limulus, a sample was withdrawn anaerobically, and the p¥L was measured at
a given temperature. The temperature of
the microelectrode was then adjusted to a
new temperature and the pH measurements were repeated. Values were obtained
at 10, 20, and 30 C.
Bicarbonate concentrations were calculated from the mean pH and PCOo of
each group of animals using the Hender-
559
INVERTEBRATE ACID-BASE REGULATION
TABLE 1. pK, Pcoi, and RCOf values as a function of temperature—Crustacea.
Water-breathing
Temp °C
Carcinus
5
10
15
20
pH
8.07 -i- .05
(11)
7.86 •+- .04
7.79 -i- .02
(5)
(6)
7.77 -1- .01
(8)
2.3
4.4 -t- .5
Pco.
mm Hg
HCO,"
raM/1
•+• . 2
4.7
•+• . 5
4.2 -1- .3
(8)
(4)
W
(7)
16.7
16.0
13.2
10.2
10
15
20
8.03 •+• 0.01
7.80 -f- 0.2
7.77 •+- 0.01
(8)
(4)
PcOo
mm Hg
HCO S nill/i
3.5 -+• 0.2
3.2 -4- 0.8
(8)
(4)
9.2
Temp °C
Occarciniis
15
Temp °C
Callinectes
pH
18.8
(8)
2.4
•+- 0.1
(8)
5.9
Air-breathing
21
30
pH
7 . 5 0 •+•
Pco2
mm Hg
(11)
8.0 -4-0.7
(11)
11.5
7.49 -i- .01
(15)
8.9 -1- 0.7
(12)
11.4
7.38 -»- .02
(14)
8.7 -4- 1.2
(3)
7.6
10
20
30
7.89 -1- .02
7.76 -t- .02
(8)
(5)
5.1 -1- 0.2
9.7 -4- 0.5
7.49 -1- .05
(6)
16.9 -1- 2.5
(6)
.19.2
HCO3-
.01
32
7.34 -t- .04
(5)
10.4 -4- 1.5
(3)
8.5
raM/l
Tomp °C
XJca
pH
Pco2
HC03"
(8)
(6)
21.1
23.2
111M/!
Mean values for pH and P c o , ± standard error. Values in parentheses represent the number
of animals. HCO3 was calculated from the average pH and PCo2 values at each temperature.
son-Hasselbalch equation (pH = pK -\- log
(HCO3-) \
1 . Since neither the pK. nor the
aPco
/
COL, solubility is known for hemolymph, we
chose to use the values given by Truchot
(1973) in order to be able to compare our
data with those he reported for Carcinus.
RESULTS AND DISCUSSION
The in vivo pH, PC02, and HCO 3 ~
values for four crab species are presented
in Table 1. Figure 2 shows the mean pH
values of these four species and also the
king crab Parolithod.es as a function of
temperature. The A/>H/A°C for all of the
crabs is very similar and appears to be
essentially the sarnie as AjWV/A°C.
Although both for Carcinus (Truchot,
1973) and for Callinectes and Limulus
(Mangum, 1973) lower absolute pli values
than those found in our animals are reported, the A/>H/A°C that Truchot found
for Carcinus in vivo is —.0162, the same
slope that we find for Carcinus. The difference in the absolute pH values may be a
result of the methods used in obtaining or
handling the hemolymph but is more likely
accounted for by the environment in which
the animals were maintained. All of the
560
HOWELL, RAHN, GOODFELLOW, AND HERREID
animals in question were kept in sea water,
and the pH of water-breathers is known
to be affected by alterations in factors such
as ionic content and P<x>2 i n their respiratory medium (Dejours et al., 1968). The
interesting observation here is that, regardless of what the pH-pN is in a given
group of animals, they maintain that degree of relative alkalinity as their temperature changes.
In Figure 3 we have plotted the average
pH values of all the crabs studied as a
function of the temperature. The bottom
diagonal line is pN and each line above
that represents a particular pFL-pN or
OH/H ratio. The OH/H ratio is simply
another way of describing relative alkalinity
and is equal to the antilog of 2(pH-pN).
The OH/H ratio for water is 1.0 at all
temperatures; thus, the higher the OH/H
ratio is for an animal, the greater is its
relative alkalinity. It may be seen from this
graph that the OH/H ratio is not the same
for all species but appears to be relatively
constant for any one species. This same
phenomenon was observed in the vertebrates (Howell et al., 1970). The value for
Gecarcinus, an OH/H ratio of approximately 8, is the lowest relative alkalinity
7.0-
30
FIG. 2. The bottom line (pX) represents the pH
or pOH o£ water as a function of temperature.
The pH values for temperature-acclimated animals
repiesent the group mean.
pH
8.0
7.8
7.6
74
7.2
7.0
6.8
o Callinectes
& Carcinus
• King Crab
i="UCA
A Gecarcinus
-I
1-
-I
15
1
1
25
1 135
FIG. 3. Average pH values of the Crustacea indicating the degree of relative alkalinity (p¥L-pN)
maintained by each species. The (OH/FT) ratio is
the antilog of
we have seen, although Truchot (1973) reports an OH/H ratio of 12 for his Carcinus
and data from Mangum (1973) suggest an
OH/H ratio of approximately 4 for the
annelid Glycera dibranchiala and 6.5 for
the sipunculid Phascolopis gouldi.
Both in vivo and in vitro pH values for
the hemolymph of Limulus are given in
Table 2 and plotted in Figure 4. It is
apparent that this animal also maintains a
constant relative alkalinity. The same
ApH/A°C is obtained in vivo and in vitro.
This same phenomenon occurs in the frog
and turtle (Howell et al., 1970) and in
Carcinus maenas (Truchot, 1973), and
strongly suggests that the CO2 content of
the animal's blood is held constant as the
temperature changes. Reeves (1972) has
shown by direct measurement that the CO2
content of frog blood is constant, but
Truchot (1973), using calculated total CO2
content, argues that the CO2 content in
Carcinus is not constant. Regardless of the
mechanism, it certainly appears that the
invertebrates, like the vertebrates, regulate
INVERTEBRATE ACID-BASE REGULATION
561
TABLE 2. pH, I'cOt, and HCOS~ values as a function of temperature—Liimilus polyphemus.
10
Water-breathing
15
7.66 •+- .05
(11)
3.8 -+- 1.3
(11)
8.6
7.62 -*- .01
(8)
4.3 -1- 0.6
(8)
8.0
Temp °C
20
30
7.48 -1- .02
(19)
5.0 -+• 0.7
(21)
7.27 -+• .02
(11)
5.5 -1- 0.4
(9)
3.9
In vivo
Pn
Pco 2
mm Hg
HCO3mM/i
In vitro
pH
7.0
7.65 -+- .02
(6)
4.5 -+- 0.1
(5)
10.7
Pco 2
mmHg
HCO3mU/l
7.49 •+- .01
(6)
4.8 •+• 0.3
7.31 •+• .03
(6)
6.4
(6)
6.1
(6)
8.1
•+• 0.5
Mean values for pH aaid PCo2 ± standard error. Values in parentheses represent the number
of animals. HCO3 was calculated from the average pH and Pro, values at each temperature.
their extracellular fluid pH in such a way
that they, too, maintain a fairly constant
relative alkalinity.
It is interesting to speculate on the
mechanisms which these animals use to
regulate the pH of their extracellular fluid.
As Henderson (1908) pointed out many
years ago, the most efficient and rapid
method for regulating pH is by altering
PC02 via ventilation and HCO 3 - by a
renal mechanism. Thus, an animal exposed
to increasing temperature could permit its
pH
7.6
^^
^ ^ i n Vitro
74
7.2
7.0
Limulus
-1
10
-120
30
FIG. 4. The solid line represents the mean in vitro
pH values and the open circles represent the mean
in vivo pH values of Limulus hemolymph as a
function of temperature.
PC02 to rise while maintaining its bicarbonate constant or permit the HCO3~ to
fall while holding PC02 at a fixed value or
utilize a combination of the two.
The data in Tables I and 2 show that
the Pco2 °f 'he water-breathing animals
at any temperature is considerably less
than the PCo2 °f the air breathers. Rahn
(1966a) showed theoretically that the PC02
of water-breathing fish and amphibians
could not rise above 5 mm Hg even if the
animal extracted all of the oxygen from
the water. The reason for this limitation
in aquatic animals is to be found in the
different solubilities of O2 and CO2 in
water.
In air the partial pressure of any gas
is proportional to the fractional volume
which the gas occupies, PG cc (PB • FG).
Thus a milliliter of O2 would exert the
same pressure as a milliliter of CO2 or No.
If iJ = ], Pi = 150 mm Hg, and
?
Pi
were negligible, extraction of all
co2
of the oxygen passing over the respiratory
surface of an animal would result in a
PE
of 150 mm Hg. In water, however,
2
the partial pressure of a gas is determined
by both fractional volume and the soluP *F
bility of the gas, PG cc ———, thus the
greater the solubility, the less will be the
562
HOWELL, RAHN, COODFELLOVV, AND HERREID
partial pressure exerted by a given gas
volume. CO2 is far more soluble in water
than O2 at any temperature and at 10 C
is about 30 times more soluble. Thus, an
animal breathing water at 10 C, under the
same circumstances we set for our airbreather, would have a maximum P B
of 5 mm Hg if it extracted all of the oxygen
from the water passing over its respiratory
surface. This upper limit, 5 mm Hg,
applies to animals living in fresh water.
Dejours et al. (1968) suggest that in sea
water, with its high HCO 3 ~, the upper
may actually be less than
limit for P B
5 mm Hg.
Rahn and Baumgardner (1972) have discussed the limitations placed on waterbreathing fish by the characteristics of their
respiratory medium and have pointed out
that if an aquatic animal is to exceed the
PCo2 ceiling it must do so by breathing
air. The gar pike, Lepisosteous, is a waterbreather at low temperatures and has a
PC02 of about 3 mm Hg at 10 C, but at
higher temperatures the gar pike, a facultative air-breather, supplies its oxygen needs
by breathing air, and at 30 C its PC02 is
13 mm Hg (Rahn et al., 1971).
In fish which are obligatory airbreathers such as Electrophorus (Garey and
Rahn, 1970) and the lungfish Protoptenis
(Lenfant and Johansen, 1968; Lahiri et
al., 1970), the PC02 values are approximately six times those found in waterbreathing fish. In amphibians, Erasmus
et al. (1970) have shown that the adult
bullfrog Rana catesbeiana and the bullfrog tadpole have the same pH at a given
temperature, but the P c o , of the adult frog
at 20 C is 13 mm Hg compared to 3.2
mm Hg for the water-breathing tadpole.
The increase in PCO2 t r i at occurs as vertebrates move from water to air is fairly well
documented (Howell, 1971), and it appears
that the same event occurs in the invertebrates. Figure 5 is a bar graph of the P c02
at 15 C of the invertebrates we have
studied. The wavy line at Pco- — 5 mm
Hg represents the theoretical limit imposed
on water-breathing animals. It must be
2h
King Crab Collinectes Limulus Corcinus UCA Gecorcinus
FIG. 5. Average hemolymph P c o values of animals acclimated to 15 C. Values below the wavy
line at P c o = 5 are from animals breathing water
while those above the line are from air-breathing
animals.
pointed out that these values were not
obtained on "arterial" or just post-gill
blood and that they probably represent
venous values, which would be expected
to be a little higher than arterial Poo
values. Nevertheless, it is apparent that
the P 002 of water-breathers is less than
that of the air breathers.
Our data for Callinectes, Garcinus, and
Limulus (Tables 1 and 2) tend to confirm
the PCO2 limit for aquatic animals and
suggest that regulation of pH in these
animals is achieved by manipulation of
the HCO3~ when the limiting P c02 is
approached. A comparison of the in vitro
and in vivo Limulus data is particularly
interesting. At 10 and 20 C the in vivo and
in vitro Pcoa a n ( i HCO 3 ~ are essentially
the same and are at the theoretical limit
for PC02. At 30 C, the in vitro P co= is 8
and the bicarbonate has changed very little
from the 20 C value, while the in vivo
Pco2 is 5.5 mm of Hg and the HCO3~ is
about 50% of the 20 C value, indicating
that the appropriate pH is achieved in the
living animal by reduction of HCO3~
whereas in the non-regulated in vitro
system, a rise in P c o , is the cause of the
fall in pH.
In the air-breathers, Uca and Gecarcinus,
the decrease in HCO a - with increasing
INVERTEBRATE ACID-BASE REGULATION
temperature is far less striking: indeed one
might suggest that in Uca the bicarbonate
is constant and that they, free of the CO2
lid, regulate pH primarily by changing
Gecarcinus, on the other hand, show a
small increase in PC02 between 15 and
32 C and a small decrease in HCO3~ suggesting that they regulate their pH by a
combination of ventilatory and renal
mechanisms.
These observations suggest that regulation of the relative alkalinity of the extracellular fluid, and the mechanisms used to
preserve this alkalinity are similar in
vertebrates and invertebrates.
REFERENCES
Astrup, P. 1956. A simple electrometric technique
for the determination of carbon dioxide tension
in blood and plasma, total content of carbon
dioxide in plasma, and bicarbonate content in
"separated" plasma at a fixed carbon dioxide tension (40 mm Hg) . Scand. J. Clin. Invest. 8:33-43.
Austin, J. H., and G. C. Cullen. 1925. Hydrogen
ion concentration of the blood in health and disease. Medicine 4:275-343.
Austin, J. H., F. W. Sunderman, and J. G. Camack.
1927. Studies in serum electrolytes. II. The electrolyte composition and the pH of serum of a
poikilothermous animal at different temperatures. J. Biol. Chem. 72:677-685.
Baumgardner, F. W. 1971. Acid-base balance in vertebrates. Ph.D. Dissertation, State University of
New York at Buffalo, N.Y.
Dejours, P., J. Armand, and G. Verriest. 1968. Carbon dioxide dissociation curves of water and gas
exchange of water breathers. Resp. Physiol. 5:2333.
Erasmus, B. DeW., B. J. Hovvell, and H. Rahn.
1970. Ontogeny of acid-base balance in the bullfrog and chicken. Resp. Physiol. 11:46-53.
563
Garey, W. F., and H. Rahn. 1970. Normal arterial
gas tensions and pH and the breathing frequency of the electric eel. Resp. Physiol. 9:141-150.
Henderson, L. J. 1908. The theory of neutrality
regulation in the animal organism. Amer. J.
Physiol. 21:427-448.
Howell, B. J. 1970. Acid-base balance in transition
from water breathing to air breathing. Fed. Proc.
29:1130-1134.
Howell, B. J., F. W. Baumgardner, K. Bondi, and
H. Rahn. 1970. Acid-base balance in cold-blooded
vertebrates as a function of body temperature.
Amer. J. Physiol. 218:600-606.
Lahiri, S., J. P. Szidon, and A. P. Fishman. 1970.
Potential respiratory and circulatory adjustments
to hypoxia in the African lungfish. Fed. Proc.
29:1141-1148.
Lenfant, C, and K. Johansen. 1968. Respiration in
the African lungfish Protopterus aethiopicus. J.
Exp. Biol. 49:437-452.
Mangum, C. P. 1973. Evaluation of the functional
properties of invertebrate hemoglobin. Neth. J.
Sea Res. 6. (In press)
Rahn, H. 1966a. Aquatic gas exchange: theory.
Resp. Physiol. 1:1-12.
Rahn, H. 19666. Gas transport from the external
environment to the cell, p. 3-23. In A. V. S. de
Reuck and R. Porter [ed.], Development of the
lung. J. & A. Churchill Ltd., London.
Rahn, H. 1971. Aoid-base regulation and temperature in the evolution of vertebrates. Proc. Int.
Union Physiol. Sci. 8:91-92.
Rahn, H., K. B. Rahn, B. J. Howell, C. Gans, and
S. M. Tenney. 1971. Air breathing of the garfish (Lepisosteus osseus) . Resp. Physiol. 11:285307.
Rahn, H., and F. W. Baumgardner. 1972. Temperature and acid-base regulation in fish. Resp.
Physiol. 14:171-182.
Reeves, R. B. 1972. An imidazole alphastat hypothesis for vertebrate acid-base regulation: tissue carbon dioxide content and body temperature in bullfrogs. Resp. Physiol. 14:219-236.
Truchot, J. P. 1973. Temperature and acid-base
regulation in the shore crab Carcinus maenas
(L). Resp. Physiol. 17:11-20.